The long term objective of this research is to understand the role of protein kinases in the regulation of smooth muscle contraction. A number of reports have suggested the effects of various protein kinases on the function of various proteins involved in stimulation-contraction coupling of smooth muscle cells. In this proposal, we focus our attention on the three protein kinases, protein kinase C (PKC) and calmodulin dependent protein kinase II (CaMPKII). While a bulk of reports have suggested that both PKC and CaMPKII play a role in the modulation of smooth muscle contractile response, it is unclear how they modulates contraction in intact cells, It may act on Ca2+ regulating proteins to decrease cytosolic Ca2+ or directly influence the regulatory proteins of myosin phosphorylation such as myosin light chain phosphatase. The complexity in identifying the role of protein kinases in smooth muscle regulation is at least partly due to the fast that there are multiple isoforms expressed in the cells. We hypothesize that each kinase isoform has a specific target site and that this is reflected by specific subcellular localization and translocation after the stimulation. The proposed project will address this problem by using ultrafast 3D digital imaging microscope (UFM) developed by us. Once we identify the subcellular localization of PKC/CaMPKII/PKG isoforms, we will study the function of these protein kinases on the Ca2+ signaling and contraction of smooth muscle cells. The effect of the kinases on contractility will be monitored by force measurement as well as single cell shortening velocity. The role of the kinases on Ca2+ homeostasis will be primarily studied by Ca2+ imaging of single cells loaded by various Ca2+ indicators using UFM. To clarify the role of protein kinases on smooth muscle contraction, we will employ genetic manipulation using cultured smooth muscle cells showing contractile phenotype, as well as by use of biochemical probes such as caged peptides, constitutively active kinases etc using freshly isolated strips/cells. The localization/translocation data will be utilized for the use of probes and the design of experiments. The following itemized specific aims and experiments will be carries out. Specific aim 1. To define the mechanism and signals which initiate the translocation of PKCs and CaMPKII. Specific aim 2. To define the role of PKC and CaMPKII on Ca2+ signaling and contraction of smooth muscle cells. Specific aim 3. To define the protein targets phosphorylated by the PKC and CaMPKIIcascade in smooth muscle cells.